Kinase substrate sensor

ABSTRACT

The disclosure relates to a cytoplasmic protein complex comprising: (a) a first recombinant fusion protein comprising a kinase, fused to a first interaction polypeptide; and (b) a second recombinant fusion protein comprising a domain comprising a reporter phosphorylation site, whereby the domain is fused to a second interaction polypeptide. The disclosure relates further to a method to detect compound-compound-interaction using the cytoplasmic protein complex, and to cells comprising such cytoplasmic protein complex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2012/053463, filed Feb. 29, 2012,designating the United States of America and published in English asInternational Patent Publication WO 2012/117031 A1 on Sep. 7, 2012,which claims the benefit under Article 8 of the Patent CooperationTreaty and under 35 U.S.C. §119(e) to Great Britain Patent ApplicationSerial No. 1103453.5, filed Mar. 1, 2011, and to U.S. Provisional PatentApplication Ser. No. 61/464,285, filed Mar. 1, 2011.

TECHNICAL FIELD

The disclosure relates to a cytoplasmic protein complex comprising (a) afirst recombinant fusion protein comprising a kinase, fused to a firstinteraction polypeptide and (b) a second recombinant fusion proteincomprising a domain comprising a reporter phosphorylation site, whereinthe domain is fused to a second interaction polypeptide. It relatesfurther to a method of detecting compound-compound-interaction using thecytoplasmic protein complex, and to cells comprising such cytoplasmicprotein complex.

BACKGROUND

Protein-protein interactions are an essential key in all biologicalprocesses, from the replication and expression of genes to themorphogenesis of organisms. Protein-protein interactions govern, amongstothers, ligand-receptor interaction and the subsequent signalingpathway; they are important in assembly of enzyme subunits, in theformation of biological supramolecular structures such as ribosomes,filaments and virus particles, and in antigen-antibody interactions.

Researchers have developed several approaches in attempts to identifyprotein-protein interactions. Co-purification of proteins andco-immunoprecipitation were amongst the first techniques used. However,these methods are tedious and do not allow high throughput screening.Moreover, they require lysis corrupting the normal cellular context. Amajor breakthrough was obtained by the introduction of the geneticapproaches, of which the yeast two-hybrid (Fields and Song, 1989) is themost important one. Although this technique became widely used, it hasseveral drawbacks. The fusion proteins need to be translocated to thenucleus, which is not always evident. Proteins with intrinsictranscription activation properties may cause false positives. Moreover,interactions that are dependent upon secondary modifications of theprotein such as phosphorylation cannot be easily detected.

Several alternative systems have been developed to solve one or more ofthese problems.

Approaches based on phage display do avoid the nuclear translocation.WO9002809 describes how a binding protein can be displayed on thesurface of a genetic package, such as a filamentous phage, wherein thegene encoding the binding protein is packaged inside the phage. Phages,which bear the binding protein that recognizes the target molecule, areisolated and amplified. Several improvements of the phage displayapproach have been proposed, as described, e.g., in WO9220791, WO9710330and WO9732017.

However, all these methods suffer from the difficulties that areinherent at the phage display methodology: the proteins need to beexposed at the phage surface and are so exposed to an environment thatis not physiologically relevant for the in vivo interaction. Moreover,when screening a phage library, there will be a competition between thephages that results in a selection of the high affinity binders.

U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybridsystem, whereby a phage library can be screened formodification-dependent protein-protein interactions. However, thismethod relies on the co-expression of the modifying enzyme, which willexert its activity in the cytoplasm and may modify enzymes other thanthe one involved in the protein-protein interaction, which may, on itsturn, affect the viability of the host organism.

An interesting evolution is described in U.S. Pat. No. 5,776,689, by theso-called protein recruitment system. Protein-protein interactions aredetected by recruitment of a guanine nucleotide exchange factor (Sos) tothe plasma membrane, where Sos activates a Ras reporter molecule. Thisresults in the survival of the cell that otherwise would not survive inthe culture conditions used. Although this method certainly has theadvantage that the protein-protein interaction takes place underphysiological conditions in the submembranary space, it has severaldrawbacks. Modification-dependent interactions cannot be detected.Moreover, the method is using the pleiotropic Ras pathway, which maycause technical complications.

A major improvement in the detection of protein-protein interactions wasdisclosed in WO0190188, describing the so called Mappit system. Themethod, based on a cytokine receptor, allows not only a reliabledetection of protein-protein interactions in mammalian cells, but alsomodification-dependent protein interactions can be detected, as well ascomplex three-hybrid protein-protein interactions mediated by a smallcompound (Caligiuri et al., 2006). However, although very useful, thesystem is limited in sensitivity and some weak interactions cannot bedetected. Moreover, as this is a membrane-based system, nuclearinteractions are normally not detected.

There is still a need for a sensitive identification system forcompound-compound interactions that can study these interactions underphysiological conditions, with a low background and by whichmodification-dependent protein-protein interactions can be isolated.

DISCLOSURE

The disclosure, based on a mutant cytoplasmic kinase, preferably aconstitutive mutant cytoplasmic kinase, satisfies this need and providesadditional advantages as well. Whereas Mappit is using aligand-receptor-controlled inducible kinase to limit and correct for thebackground, the cytoplasmic kinase, especially the constitutivecytoplasmic kinase, is surprisingly giving a higher signal/noise ratio,allowing the detection of interactions that cannot be detected in themappit system. The high signal/noise ratio is unexpected for acytoplasmic system, as a person skilled in the art would expect thatcytoplasmic kinase can move freely in the cytoplasm, and reach itssubstrate without other recruitment signals than the kinase and thephosphorylation domain. By using a cytoplasmic system, the problem ofdetecting nuclear protein-protein interactions has been solved. Theunexpectedly high sensitivity of the present method allows detectingweak protein interactions, such as the p51/p51 interaction, which cannotbe detected by Mappit. Alternatively, a mutant kinase inducible by anexogenous small compound can be used. This approach combines theadvantages of the constitutive kinase, with the inducibility, withoutinterfering with the endogenous pathways.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Schematic representation of the cytoplasmic protein complex. Afirst interacting polypeptide (X) is fused to a constitutive kinase (K)and a second interacting polypeptide (Y) is fused to a reporterphosphorylation site (R). Interaction between the interactingpolypeptides X and Y results in the reporter phosphorylation site beingphosphorylated (P) by the constitutive kinase, leading to a detectableactivity.

FIGS. 2A and 2B: Detection of the interaction between HIV1 ReverseTranscriptase (RT) subunits in an assay variant that comprises mutantTyk2 kinase fusion proteins. FIG. 2A: Schematic overview of the assay.The first interacting polypeptide (X) is fused to the C-terminal regionof Tyk2 comprising the kinase domain, whereas the second interactingpolypeptide (Y) is fused to a fragment of gp130, which containsphosphorylation sites. When polypeptides X and Y interact, the Tyk2kinase domain phosphorylates the phosphorylation sites of gp130. STAT3transcription factors are recruited to these phosphorylated sites andare, in turn, phosphorylated by the Tyk2 kinase domain, which results intheir activation. Dimers of activated STAT3 transcription factors areable to bind the specific rPAPI promoter, which drives the expression ofa firefly luciferase reporter gene. The activity of this reporter geneis measured as light production in a luciferase detection assay using aluminometer. FIG. 2B: Application to the analysis of HIV1 RT subunits.Cells were transfected with the following plasmids: a)pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase, b)pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase, c)pMet7-HA-Tyk2(C)-RTp51+pMG2-RTp51+pXP2d2-rPAPI-luciferase. Luciferaseactivity is shown as fold induction relative to the luciferase activitymeasured in cells transfected with the same Tyk2(C) fusion, an unfusedgp130 fragment and the luciferase reporter plasmid(pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in a) and b);pMet7-HA-Tyk2(C)-RTp51+pMG1+pXP2d2-rPAPI-luciferase in c). Error barsindicate standard deviation.

FIG. 3: Detection of the interaction between HIV1 Integrase (IN) andhuman LEDGF and between human p53 and MDM2. Cells were transfected withthe following plasmids: a)pMet7-HA-Tyk2(C)-LEDGF+pMG2-IN+pXP2d2-rPAPI-luciferase and b)pMet7-HA-Tyk2(C)-MDM2+pMG1-p53+pXP2d2-rPAPI-luciferase. Luciferaseactivity is shown as fold induction relative to the luciferase activitymeasured in cells transfected with the same Tyk2(C) fusion, an unfusedgp130 fragment and the luciferase reporter plasmid(pMet7-HA-Tyk2(C)-LEDGF+pMG1+pXP2d2-rPAPI-luciferase in a);pMet7-HA-Tyk2(C)-MDM2+pMG1+pXP2d2-rPAPI-luciferase in b)). Error barsindicate standard deviation.

FIG. 4: Dose-dependent disruption of the interaction between human p53and MDM2 by Nutlin-3. Cells were transfected with the followingplasmids: a) pMet7-HA-Tyk2(C)-p53(N)+pMG1+pXP2d2-rPAPI-luciferase; b)pMet7-HA-Tyk2(C)-p53(N)+pMG1-EFHA1+pXP2d2-rPAPI-luciferase; and c)pMet7-HA-Tyk2(C)-p53(N)+pMG2-MDM2+pXP2d2-rPAPI-luciferase. Aftertransfection, cells were treated with 0-14-41-123-370-1111-3333 nMNutlin-3, final concentration. Error bars indicate standard deviation.

FIG. 5: Detection of the interaction between HIV1 Reverse Transcriptase(RT) p66 subunits in assay variants that comprise mutant Jak2 or c-Srckinase fusion proteins. Cells were transfected with the followingplasmids: a) pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase;b) pMet7-HA-Jak2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase; and c)pMet7-HA-c-Src(K)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase. Luciferaseactivity is shown as fold induction relative to the luciferase activitymeasured in cells transfected with the same RTp66-kinase fusion, anunfused gp130 fragment and the luciferase reporter plasmid(pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in a);pMet7-HA-Jak2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in b);pMet7-HA-c-Src(K)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in c)). Error barsindicate standard deviation.

FIG. 6: Detection of the interaction between HIV1 Reverse Transcriptase(RT) subunits in an inducible assay variant. Cells were transfected withthe following plasmids: a)pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase; b)pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase; c)pMet7-HA-Tyk2(R1027A)-RTp66+pMG1+pXP2d2-rPAPI-luciferase; and d)pMet7-HA-Tyk2(R1027A)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase. Aftertransfection, cells were treated with 0-5-50 mM imidazole, finalconcentration. Error bars indicate standard deviation.

DETAILED DESCRIPTION

A first aspect hereof is a cytoplasmic protein complex comprising (a) afirst recombinant fusion protein comprising a kinase fused to a firstinteraction polypeptide and (b) a second recombinant fusion proteincomprising a domain comprising a reporter phosphorylation site, whereinthe domain is fused to a second interaction polypeptide.

The first recombinant fusion protein need not comprise a reporterphosphorylation site. Preferably, the kinase is a mutant kinase, morepreferably, a mutant tyrosine kinase. In one embodiment, the mutanttyrosine kinase is a constitutive kinase. In another embodiment, themutant tyrosine kinase is an inactive mutant that is activated byaddition of an exogenous small molecule. Such mutant kinase is known tothe person skilled in the art, and has been described, as a non-limitingexample, by Qiao et al. (2006) as a Src 388R/A mutant or a 391R/Amutation in the corresponding human Src protein (Accession numberNP_(—)938033, version NP_(—)938033.1). Alternatively, it may be asimilar mutation in the Jak kinase family, such as, but not limited toTyk2 1027R/A.

In one embodiment, the kinase is a constitutive kinase mutant derivedfrom Tyk2, such as, but not limited to, a constitutive Tyk2 deletionmutant and/or a Tyk2 V678F mutant. “Derived from Tyk2” as used hereinmeans that the kinase is a part of the human Tyk2 non-receptortyrosine-protein kinase (Genbank accession number NP_(—)003322; versionNP_(—)003322.3; SEQ ID NO:26) or a mutant or variant thereof, whereinthe part shows constitutive kinase activity. A variant, as anon-limiting example, is a homologue, paralogue or orthologue.“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived. “Orthologues” and“paralogues” encompass evolutionary concepts used to describe theancestral relationships of genes. “Paralogues” are genes within the samespecies that have originated through duplication of an ancestral gene;“orthologues” are genes from different organisms that have originatedthrough speciation, and are also derived from a common ancestral gene.Preferably, the homologue, “orthologue” or “paralogue” has a sequenceidentity at protein level of at least 50%, 51%, 52%, 53%, 54% or 55%,56%, 57%, 58%, 59%, preferably 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, more preferably 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, even more preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, and most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more as measured in a BLASTp (Altschul et al., 1997; Altschul etal., 2005). Variants and parts thereof according to the invention doshow kinase activity. Preferably, the part is a part with constitutivekinase activity, preferably fragment 589-1187 of SEQ ID NO:26.Alternatively, the part is the part corresponding to fragment 589-1187of SEQ ID NO:26 in a homologue, paralogue or orthologue as definedabove, wherein the part has constitutive kinase activity. In analternative embodiment, the constitutive kinase is a constitutive kinasederived from a Jak kinase, preferably from a Jak kinase selected fromthe group consisting of Jak1 (Accession number P23458, versionP23458.2), Jak2 (Accession number 060674, version 060674.2), and Jak3(Accession number P52333, version P52333.2), or a mutant or variantthereof as defined above. Preferably, the constitutive kinase is aconstitutive Jak2 deletion mutant. In still another alternativeembodiment, the constitutive kinase is a constitutive kinase derivedfrom a Src kinase (Accession number NP_(—)005408, versionNP_(—)005408.1) or a mutant or variant thereof as defined above.Preferably, the constitutive kinase is a Src deletion mutant as depictedin SEQ ID NO:27. “Derived” as used herein means that the kinase is apart of the cited non-receptor tyrosine-protein, or from a mutant orvariant thereof, wherein the part shows constitutive kinase activity.

In one embodiment, the first recombinant fusion protein comprises SEQ IDNO:1 (pseudo kinase+kinase). In another embodiment, the firstrecombinant protein comprises SEQ ID NO:2 (kinase). In still anotherembodiment, the first recombinant protein comprises a sequence selectedfrom the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, andSEQ ID NO:27. In one embodiment, the second recombinant fusion proteincomprises SEQ ID NO:3 (STAT site). In another embodiment, the secondrecombinant fusion protein comprises SEQ ID NO:4. Most preferably, thecytoplasmic protein complex comprises a first recombinant fusion proteincomprising SED ID NO:1 or SEQ ID NO:2 and a second recombinant fusionprotein comprising SEQ ID NO:3 or SEQ ID NO:4.

In cases where an inactive mutant is used that can be activated by asmall compound, one preferred embodiment is a mutant kinase whereby themutant kinase is a Src 388R/A mutation or a 391R/A mutation in thecorresponding human Src protein (Accession number NP_(—)938033, versionNP_(—)938033.1). Another embodiment is a mutant wherein the mutantkinase is a Tyk2 1027R/A mutant (numbering of SEQ ID NO:26). Preferably,the small compound is imidazole.

The kinase of the first recombinant fusion protein is fused to a firstinteraction polypeptide, as defined below. The fusion may occur at theamino-terminal end, at the carboxy-terminal end, or internal in therecombinant polypeptide. In cases of kinases consisting of two domains,the first interaction polypeptide may even occur as an internal fusionin between the two domains. The domain comprising the reporterphosphorylation site of the second recombinant fusion protein is fusedto a second interaction polypeptide, as defined below. The fusion mayoccur at the amino-terminal end, at the carboxy-terminal end, orinternal in the recombinant polypeptide. The first recombinant fusionprotein may not comprise a reporter phosphorylation site. The firstrecombinant fusion protein, the second recombinant fusion protein orboth may further comprise other sequences such as, but not limited to, alocalization signal to direct the cytoplasmic complex to a specificorganelle into the cytoplasm of the cell, or to the nucleus.

Preferably, the cytoplasmic protein complex is phosphorylated at thereporter phosphorylation site; even more preferably, the phosphorylationis carried out by the kinase.

In an embodiment, the protein complex according to the invention isassembled by direct interaction of the first interaction polypeptidewith the second interaction polypeptide. The first and the secondinteraction protein may be identical, in cases where homodimerization isstudied. However, the protein complex is not limited to two compounds,and the interaction may be mediated by one or more other compounds. As anon-limiting example, the first interaction polypeptide may bind aprotein that is bound on its turn by the second interaction polypeptide.Instead of a protein, the bridging compound can be one or more moleculesof non-proteinous nature, or mixtures of small compounds and proteins.Another non-limiting example is a protein complex, wherein a smallcompound binding to the first, as well as to the second, interactionpolypeptide.

The complex formation may be dependent upon modification of the firstand/or second interaction polypeptide. Modification can be, but is notlimited to, presence or absence of phosphorylation, acetylation,acylation, methylation, ubiquitinilation or glycosylation, or occurrenceof proteolytic cleavage or not, or a combination thereof. Preferably,modification is carried out by a modifying enzyme (as defined below).Preferably, the modifying enzyme is fused to the first and/or secondrecombinant fusion protein. In case of phosphorylation, the modifyingenzyme may be the constitutive kinase fused to the first interactionpolypeptide, as described above.

Another aspect hereof is a method for detecting compound-compoundinteraction comprising (a) fusing a cytoplasmic kinase to a firstinteraction polypeptide resulting in a first recombinant fusion protein(without reporter phosphorylation site) according to the invention; (b)fusing a domain comprising a reporter phosphorylation site to a secondinteraction polypeptide, resulting in a second recombinant fusionprotein according to the invention; (c) expressing both recombinantfusion proteins in a cell; and (d) identifying and/or selecting thosecells in which the reporter phosphorylation site is phosphorylated.

Preferably, the kinase is a mutant kinase, even more preferably, amutant tyrosine kinase. In one embodiment, the mutant kinase is aconstitutive kinase; even more preferably, the kinase is a constitutivemutant derived from a Tyk2 kinase, preferably a deletion mutant or aconstitutive V678F mutant. Alternatively, a constitutive Jak2 deletionmutant or a constitutive Src kinase deletion mutant can be used. Inanother embodiment, kinase is an inactive mutant kinase that isactivated by addition of an exogenous small compound. Such mutant kinaseis known to the person skilled in the art and has been described, as anon-limiting example, by Qiao et al. (2006).

In one embodiment, the first recombinant fusion protein comprises SEQ IDNO:1. In another embodiment, the first recombinant fusion proteincomprises SEQ ID NO:2. In still another embodiment, the firstrecombinant protein comprises a sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:27.In one preferred embodiment, the second recombinant fusion proteincomprises SEQ ID NO:3. In another preferred embodiment, the secondrecombinant fusion protein comprises SEQ ID NO:4. Most preferably, thecytoplasmic protein complex comprises a first recombinant fusion proteincomprising SED ID NO:1 or SEQ ID NO:2 and a second recombinant fusionprotein comprising SEQ ID NO:3 or SEQ ID NO:4.

The gene encoding the first and/or the second recombinant fusion proteinmay be placed downstream and is either a constitutive or an induciblepromoter. The latter construction may have some advantages in caseswhere there is a competition for the binding site between interactionpolypeptide and endogenous polypeptides. Induction of the recombinantfusion protein comprising the first interaction polypeptide in thepresence of the second interaction polypeptides may facilitate thebinding and avoid saturation of the binding sites with endogenouspolypeptides. The cell may be any cell including, but not limited to,bacterial cells, fungal cells, yeast cells, insect cells and mammaliancells. Preferably, the cell is a eukaryotic cell; even more preferably,the cell is a mammalian cell. In cases where homodimerization isstudied, the first and the second interaction protein may be identical.In this case, the same protein is fused to the kinase at one hand, andto a domain comprising the reporter phosphorylation site at the otherhand.

The first recombinant fusion protein and the second recombinant fusionprotein may be situated on one or on separated vectors. The vector usedfor transformation and expression of the first and/or second recombinantpolypeptide can be any vector known to the person skilled in the artincluding, but not limited to, episomal vectors, integrative vectors andviral vectors.

In an embodiment, a eukaryotic cell carrying a gene encoding a firstrecombinant fusion protein is transformed or transfected with a vectorlibrary encoding second recombinant fusion proteins hereof. Interactionof the first interaction polypeptide with the second interactionpolypeptide will result in phosphorylation of the reporterphosphorylation site and can be detected by the use of a reportersystem. One specific embodiment of the method to detectcompound-compound binding is a method whereby the compounds are proteinsand binding is a protein-protein interaction. Another specificembodiment is a method to detect protein-protein interaction, wherebythe interaction is modification state dependent, and modification iscarried out by a modifying enzyme. Still another specific embodiment isa method to detect compound-compound binding, whereby binding ismediated by three or more partners. In this case, one or more partnersmay not be, or not completely be, of proteinous nature. It is clear fora person skilled in the art that the first interaction polypeptide may,as a non-limiting example, bind to a small molecule. On the other hand,the second interaction polypeptide may also bind to the small molecule,so that the first and the second are linked together by the smallmolecule. The small molecule may be present in the host cell, as acompound produced by the cell itself, or as a compound that is taken upfrom the medium.

Preferably, the method to detect compound-compound binding comprises theconstruction of a eukaryotic cell comprising a first recombinant fusionprotein hereof, followed by transformation or transfection of the cellby a library of vectors encoding second recombinant fusion proteinsaccording to the invention. The compound-compound binding is detected bythe phosphorylation of the reporter phosphorylation site. Detection ofthe phosphorylation of the complex can be realized by isolation of thecomplex and analyzing the phosphorylation (e.g., by using radioactivelabeled phosphor). Preferably, phosphorylation of the reporterphosphorylation site is resulting in the induction of a reporter system.A reporter system can be any system that allows the detection and/or theselection of the cells carrying a phosphorylated cytoplasmic proteincomplex according to the invention. It is clear for the person skilledin the art that several reporter systems can be used. As a non-limitingexample, phosphorylation may lead to the induction of a signalingpathway, and a luciferase gene; an antibiotic resistance gene or a cellsurface marker gene can be placed under control of a promoter that isinduced by the signaling pathway. Alternatively, reporter systems may beused that are based on the change in characteristics of compounds of thesignaling pathway, when the pathway is active, such as thephosphorylation and/or dimerization of such compounds. Still anotherpossibility is using antibodies and/or other binding polypeptides whichspecifically recognize the phosphorylated reporter phosphorylation site.

One embodiment is a reporter phosphorylation site that is part of aSignal Transducer and Activator of Transcription (STAT) binding site,most preferentially, part of a STAT1 and/or STAT3 binding site. In thiscase, the reporter gene can be placed under the control of aSTAT-inducible promoter, such as, but not limited to, the PancreatitisAssociated Protein 1 (rPAP) promoter. Alternatively, as phosphorylationof the reporter phosphorylation site will result in binding of a STATpolypeptide to the phosphorylated reporter phosphorylation site,followed by phosphorylation of the STAT polypeptide and subsequentdimerization of two phosphorylated STAT molecules, the dimerizationitself can be used as reporter signal. Still another alternativereporter system consists of a protein complementation assay, wherein onepart of the protein is incorporated in or associated with thecytoplasmic protein complex according to the invention, and the secondpart of the protein is recruited to the phosphorylated reporterphosphorylation site, leading to a detectable activity of thereconstituted protein.

Still another aspect hereof is a cell comprising a cytoplasmic proteincomplex according to the invention. This cell may be any cell including,but not limited to, bacterial cells, fungal cells, yeast cells, insectcells and mammalian cells. Preferably, the cell is a eukaryotic cell;even more preferably, the cell is a mammalian cell. Preferably, thereporter phosphorylation site in the cytoplasmic protein complex isphosphorylated. Preferably, the cell also comprises a reporter system,allowing the detection of the phosphorylation of the reporterphosphorylation site of the cytoplasmic protein complex.

Still another aspect hereof is a method for detecting compoundsdisrupting a polypeptide-polypeptide interaction, the method comprising(a) growing a cell comprising a protein complex according to theinvention, wherein the cytoplasmic protein complex comprises thepolypeptide-polypeptide interaction that one wants to disrupt, inabsence and in presence of at least one compound; (b) comparing thephosphorylation of the reporter phosphorylation site in the secondrecombinant fusion protein of the cytoplasmic protein complex of cells,grown in presence or absence of the compound; and (c) identifying and/orselecting those cells wherein the reporter phosphorylation site is notphosphorylated. It is clear for the person skilled in the art that thepolypeptide-polypeptide interaction as mentioned above may be anindirect interaction by three of more partners, whereby even one or moreof the partners may not be of proteinous nature.

DEFINITIONS

The following definitions are set forth to illustrate and define themeaning and scope of various terms used to describe the inventionherein.

“Protein” as used herein means a chain composed of amino acids,independent of the length. The terms “protein” and “polypeptide” areinterchangeable. The protein can be modified by modifications such as,but not limited to, phosphorylation, glycosylation, ubiquitinilation andacetylation.

“Domain” as used herein is a part of a polypeptide, wherein the part maycarry a specific function, such as, but not limited to, an enzymaticcenter or a phosphorylation site.

“Protein complex” as used herein means a structure that comprises atleast two, non-covalently linked, protein molecules. Protein complexescan consist of more than two proteins and include other molecules thatare not proteins. Some non-limiting examples of such molecules are metalions, ATP, or carbohydrate molecules.

“Cytoplasmic protein complex” means that the protein complex asdescribed above moves freely in the cytoplasm of the cell and is notlinked to the cell membrane. However, the cytoplasmic protein complexmay be directed to the nucleus or cytoplasmic organelles, usinglocalization signal sequences.

A “kinase” as used herein is a polypeptide that can transfer a phosphategroup to an amino acid of the same or another polypeptide. Preferably,the amino acid is a serine, a threonine or a tyrosine. Even morepreferably, the amino acid is embedded in a phosphorylation site.

A “phosphorylation site” as used herein is a pattern of several aminoacids, preferably comprising a serine, threonine or a tyrosine, anddetermining the amino acid that will be phosphorylated by the kinase.Most kinases can occur in an inactive state and/or in an active state,wherein the reporter phosphorylation site is only phosphorylated in theactive state of the kinase. Kinases can be switched from the inactiveform to the active form by phosphorylation, or by other modifications,such as proteolysis, or by mutation. The phosphorylation can beautophosphorylation, cross-phosphorylation (by a protein complex ofidentical kinases) or by action of another kinase.

A “cytoplasmic kinase” is a kinase that is freely moving in thecytoplasm, and not linked or recruited to the cellular membrane. Thecytoplasmic kinase according to the invention may be derived from amembrane-linked kinase, by deleting the membrane anchoring or membranerecruitment domain, without loss of the enzymatic kinase activity.

A “mutant kinase” is a kinase of which the sequence differs from thesequence that occurs normally in nature, by the replacement, deletion orinsertion of one or more amino acids. Homologues, orthologues andparalogues are not considered as mutants, as they are present as anormal form in nature.

“Constitutive” as used herein means that the kinase is continuously inthe active state, normally as a consequence of a mutation, or byproteolytic cleavage removing an inhibitor. Constitutive kinases areknown to the person skilled in the art and comprise, but are not limitedto, truncated forms of Tyk2, truncated forms of Src kinase and pointmutations such as Tyk2 (V678F), Jak1 (V658F) and Jak2(V617F).

An “inactive kinase mutant” means that that the mutant form shows akinase activity that is significantly lower than the originalnon-mutated form. Preferably, the remaining activity is lower than 50%of the original activity, more preferably lower than 20%, even morepreferably lower than 10%, and most preferably lower than 5% of theoriginal activity.

“Activated by the addition of an exogenous small compound” as usedherein means that the activity of the inactive kinase is partly ortotally restored by addition of a small compound to the cells, wherebythe small compound, exogenous to the cell, is taken up by the cell andactivates the kinase as an intracellular exogenous compound. “Activatedby the addition of an exogenous small compound” is used to make adistinction with ligand-receptor-induced activation, where a ligand isbinding to the extracellular part of a receptor, and induces activationof the kinase.

“Exogenous” as used herein means that the compound is normally notpresent in the cell.

“Reporter phosphorylation site” is the site that is phosphorylated inthe protein complex upon interaction of the first and the secondinteraction polypeptide; it is distinct from a possible phosphorylationsite in the kinase domain that is autophosphorylated in the constitutivekinase.

“First interaction polypeptide” as used herein is a polypeptide of whichone wants to study the interaction with one or more compounds. The firstinteraction polypeptide is normally referred to as a “bait” in thetwo-hybrid terminology.

“Second interaction polypeptide” as used herein is a polypeptide that ispresented to study its interaction with the first interactionpolypeptide. The second interaction polypeptide is normally referred toas a “prey” in the two-hybrid terminology. It is clear for the personskilled in the art that the first and the second interaction polypeptideare interchangeable in the invention, in this respect that either a“bait” or “prey” may be fused to constitutive kinase hereof. Indeed, theresulting protein complex will have an identical overall composition,composed of the four essential elements (first interaction polypeptide,second interaction polypeptide, constitutive kinase and reporterphosphorylation site), and independent whether the first interactionpolypeptide is fused to the constitutive kinase or the reporterphosphorylation site (wherein the second interaction polypeptide is thenfused to the reporter phosphorylation site and the constitutive kinase,respectively), the interaction of the first with the second interactingpolypeptide will lead to the formation of a cytoplasmic protein complexaccording to the invention, and will result in the phosphorylation ofthe reporter phosphorylation site. In one preferred embodiment, thefirst and the second interaction proteins are identical to studyhomodimerization or homomultimerization of a protein. In anotherpreferred embodiment, the first and the second proteins are different,allowing the study of protein-protein interactions of heterodimers orheteromultimers.

“Modifying enzyme” as used herein means any enzymatic activity thatresults in a modification of the first and/or second interactionpolypeptide. Such modification can be, as a non-limiting example,phosphorylation, acetylation, acylation, methylation, ubiquitinilationor glycosylation, occurrence of proteolytic cleavage, or a combinationthereof. Preferably, the modification is phosphorylation. The modifyingenzyme may be associated to or incorporated in the first and/or thesecond recombinant fusion protein of the cytoplasmic protein complex. Itmay be co-expressed, with the first and/or second interactionpolypeptide, or it can be fused to one or both of the polypeptides. Incase of phosphorylation, the constitutive kinase of the cytoplasmicprotein complex according to the invention may carry out both thephosphorylation of the activation site as well as the phosphorylation ofone or both of the interaction polypeptides.

“Compound” means any chemical or biological compound, including simpleor complex organic or inorganic molecules, peptides, peptido-mimetics,proteins, antibodies, carbohydrates, nucleic acids or derivativesthereof.

“Interaction” means any interaction, be it direct or indirect. A directinteraction implies a contact between the interaction partners. Anindirect interaction means any interaction whereby the interactionpartners interact in a complex of more than two compounds. Thisinteraction can be completely indirect, with the help of one or morebridging compounds, or partly indirect, where there is still a directcontact that is stabilized by the interaction of one or more compounds.

EXAMPLES Materials and Methods to the Invention Plasmids Used in theExamples

A first type of plasmids express chimeric proteins consisting of anHA-tagged C-terminal portion of human Tyk2 fused at its C-terminus tothe first interacting polypeptide and are generated in the pMet7 vector,which contains a strong constitutive hybrid SRa promoter (Takebe et al.,1988). To generate the pMet7-HA-Tyk2(C)-RTp66 plasmid, the sequenceencoding the C-terminal end of human Tyk2 comprising the kinase domain(starting from amino acids 589 and omitting the stop codon) wasamplified by PCR on cDNA from HEK293 cells with primers MBU-O-6486 andMBU-O-6487. In addition to an HA coding sequence, the former primercontained an ApaI restriction enzyme recognition site, whereas thelatter primer contained an EcoRI restriction enzyme recognition site.The PCR amplicon was digested with ApaI and EcoRI and ligated in theApaI-EcoRI cut pMG2-RTp66 plasmid (a pMet7-derived plasmid encoding aFlag-gp130-RTp66 chimeric protein), which contained ApaI and EcoRI sitesflanking the sequences encoding the Flag-gp130 fusion protein at the 5′and 3′ end, respectively (Pattyn et al., 2008). pMet7-HA-Tyk2(C)-RTp51was generated by exchanging the RTp51 insert from pMG2-RTp51 (Pattyn etal., 2008) with RTp66 from pMet7-HA-Tyk2(C)-RTp66 using the EcoRI andNotI sites that flank these inserts at the 5′ and 3′ end, respectively.Full length human LEDGF was PCR amplified on cDNA from HeLa cells usingprimers MBU-O-3879 and MBU-O-3880 and exchanged with the RTp66 insert ofpMet7-HA-Tyk2(C)-RTp66 using EcoRI and NotI restriction sites togenerate pMet7-HA-Tyk2(C)-LEDGF. Similarly, the full-length human MDM2sequence was PCR amplified on an MDM2 entry clone from the hORFeomecollection (Lamesch et al., 2007) with primers MBU-O-3912 and MBU-O-3913and exchanged with EcoRI-NotI to produce pMet7-HA-Tyk2(C)-MDM2. TheN-terminal region of human p53 (amino acids 2-72) was PCR amplified on ap53 entry clone from the hORFeome collection (Lamesch et al., 2007) withprimers MBU-O-2277 and MBU-O-2273 and exchanged using EcoRI and NotIrestriction enzymes, yielding pMet7-HA-Tyk2(C)-p53(N). The plasmidpMet7-HA-Jak2(C)-RTp66 was generated by PCR amplifying the sequenceencoding the C-terminal end of mouse Jak2 (from amino acid 535 until theend of the protein, leaving the stop codon out) on pRK5-mJak2(Silvennoinen et al., 1993) with primers MBU-O-6653 and MBU-O-6655. Thisfragment was exchanged with the sequence encoding Tyk2(C) frompMet7-HA-Tyk2(C)-RTp66 through a PacI-EcoRI restriction digest.Likewise, the coding sequence for the kinase domain of c-Src (aminoacids 266-523) was PCR amplified on a c-Src entry clone from thehORFeome collection (Lamesch et al., 2007) with primers MBU-O-6656 andMBU-O-6657 and exchanged using PacI and EcoRI restriction enzymes,generating pMet7-HA-c-Src(K)-RTp66. The pMet7-HA-Tyk2(R1027A)-RTp66plasmid was generated by first PCR amplifying the human Tyk2 sequence onHEK293 cDNA with primers MBU-O-7811 and MBU-O-7812 and exchanging theresulting amplicon for the Tyk2(C) sequence of pMet7-HA-Tyk2(C)-RTp66using PacI and EcoRI restriction enzymes to yield pMet7-HA-Tyk2-RTp66.Next, the R1027A mutation was introduced by site-directed mutagenesisusing primers MBU-O-7341 and MBU-O-7342.

The plasmids encoding the fusions with the second interactingpolypeptide were of the type also used in MAPPIT, designated pMG1 andpMG2 (WO0190188, Eyckerman et al., 2001; Lemmens et al., 2003). Theseplasmids encode fusion proteins of the second interacting polypeptidecoupled to a fragment of the human gp130 cytokine receptor chain, whichcontains multiple tyrosine residues that, upon phosphorylation, make uprecruitment sites for STAT3. RTp66 and RTp51 containing plasmidspMG2-RTp66 and pMG2-RTp51 have been described elsewhere (Pattyn et al.,2008). The pMG1 plasmid encoding an unfused gp130 receptor fragment wasobtained by cutting out the MAPPIT prey insert of a pMG1 vector usingEcoRI and XhoI, blunting the vector backbone through Pfu DNA Polymeraseand self-ligation. The pMG2-IN plasmid was constructed by PCR amplifyingthe sequence encoding HIV1 integrase on the pNL4-3 plasmid template(Adachi et al., 1986) using primers MBU-O-3813 and MBU-O-3814 andexchanging this sequence with the insert of a pMG2 MAPPIT vector usingEcoRI and NotI restriction enzymes. Similarly, the coding region ofhuman MDM2 was PCR amplified with primers MBU-O-3912 and MBU-O-3913 andexchanged with EcoRI-NotI to produce pMG2-MDM2. pMG1-p53 and pMG1-EFHA1were generated by Gateway recombination mediated transfer of thefull-length sequences of human p53 and EFHA1 from entry vectors of thehORFeome collection (Lamesch et al., 2007) into a Gateway-compatibleversion of the pMG1 vector as described earlier (Lievens et al., 2009).

The reporter plasmid pXP2d2-rPAPI-luciferase used in the examplescontains the STAT3-dependent rPAPI (rat Pancreatitis-Associated ProteinI) promoter driving a firefly luciferase reporter gene as describedpreviously (WO0190188, Eyckerman et al., 2001).

Transfection Procedure

Transfections were carried out using a standard calcium phosphatemethod. HEK293-T cells were seeded in black tissue-culture treated96-well plates at 10,000 cells/well in 100 μl culture medium (DMEMsupplemented with 10% FCS). Twenty-four hours later, plasmid DNA mixeswere prepared that contained plasmids encoding fusion proteins with thefirst and second interacting proteins and reporter plasmids. The DNA wassupplemented with 10 μl 2.5M CaCl₂ and double distilled water to a finalvolume of 100 μl. This mixture was added dropwise to 100 μl 2×HeBSbuffer (280 mM NaCl, 1.5 mM Na₂HPO₄, 50 mM Hepes; pH 7.05) whilevigorously vortexing. After incubation at room temperature for 15minutes to allow DNA precipitates to form, the solution was added to thecells at 10 μl/well. Cells were incubated at 37° C., 8% CO₂. Forty-eighthours after transfection, luciferase activity was measured using theLuciferase Assay System kit (Promega) on a TopCount luminometer(Perkin-Elmer). Each transfection was done in triplicate and the averageof the luciferase activity readings was used in the calculations.

A Nutlin-3 (Sigma) stock solution of 20 mM in DMSO was diluted inculture medium and added to the cells 24 hours after transfection.Imidazole (Sigma) diluted in culture medium was added to the cells 24hours after transfection.

Oligonucleotide SEQ ID primer code NO: Sequence (5′ > 3′) MBU-O-6486 8CCCGGGCCCACCATGTATCCATATGATGTTCCAGATTATGCTTTAATTAAAATCACCCAGCTGTCCCACTTGG MBU-O-6487 9GGGGAATTCGCACACGCTGAACACTGAAGG MBU-O-3879 10CGTACGAATTCGGGAGCTCGATGACTCGCGATTTCAA ACCTGGAG MBU-O-3880 11GGTCATCTAGACCGCGGCCGCTCAGTTATCTAGTGTA GAATCCTTCAG MBU-O-3912 12GCGGAATTCATGTGCAATACCAACATGTCTG MBU-O-3913 13CGCGCGGCCGCCTAGGGGAAATAAGTTAGCAC MBU-O-2277 14GCGAGAATTCGAGGAGCCGCAGTCAGATCC MBU-O-2273 15CGCTGCGGCCGCTTAGCGGGGAGCAGCCTCTGGC MBU-O-6653 16CCCGCGGCCGCTTTAATTAAAATGGTGTTTCACAAAA TCAG MBU-O-6655 17GGGCTCGAGGAATTCCGCAGCTATACTGTCCCGG MBU-O-6656 18CCCGCGGCCGCTTTAATTAAACCTCGGGAGTCGCTGC GGC MBU-O-6657 19GGGCTCGAGGAATTCGAAGTAGTCCTCCAGGAAGG MBU-O-3813 20CGTACGAATTCGGGAGCTCGTTTTTAGATGGAATAG MBU-O-3814 21GGTCATCTAGACCGCGGCCGCTCAATCCTCATCCTGT CTAC MBU-O-7811 22CATTTAATTAAACCTCTGCGCCACTGGGGG MBU-O-7812 23CATGAATTCGCACACGCTGAACACTGAAGGG MBU-O-7341 24CCGAGACCTAGCCGCGGCCAACGTGCTGC MBU-O-7342 25GCAGCACGTTGGCCGCGGCTAGGTCTCGG

Example 1 Detection of the Interaction Between HIV1 ReverseTranscriptase (RT) Subunits

In order to determine the functionality of the assay, the interactionbetween HIV1 subunits that form homo- and heterodimers was tested bytransfecting the following combinations of plasmids (100 ng of theTyk2(C) fusion construct, 1 μg of the gp130 fusion construct and 50 ngof the luciferase reporter construct) according to the methods describedabove:

-   -   a) pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase    -   b) pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase    -   c) pMet7-HA-Tyk2(C)-RTp51+pMG2-RTp51+pXP2d2-rPAPI-luciferase

Background signal for each Tyk2(C) fusion polypeptide was determined bytransfecting the plasmid that encodes it together with a plasmidencoding an unfused gp130 fragment (pMG1) and the luciferase reporterplasmid (pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in a) andb); pMet7-HA-Tyk2(C)-RTp51+pMG1+pXP2d2-rPAPI-luciferase in c)). The foldinduction for each tested interaction was calculated as the ratio of theluciferase activity measured relative to the luciferase activitymeasured for the corresponding background signal. The results (FIG. 2A)show clear signals for both RT homo- and heterodimers. The strongestsignal was that for the p66-p51 heterodimer; lower, but still robust,signals were obtained for the p66 and p51 homodimers. This trendcorresponds to the affinities measured for p66-p51, p66-p66 and p51-p51interactions in in vitro interaction assays, which were reported to be0.3 μM, 4 μM and 230 μM, respectively (Venezia et al., 2006). These dataillustrate the high sensitivity of the method.

Example 2 Interaction Between Nuclear Proteins

To determine whether the method can detect interactions between nuclearproteins, we tested the interaction between HIV 1 Integrase (IN) andhuman LEDGF and between human p53 and MDM2, proteins with a nuclearlocalization. Cells were transfected with the following combinations ofplasmids (250 ng of the Tyk2(C) fusion construct, 500 ng of the gp1 30fusion construct and 50 ng of the luciferase reporter construct)according to the methods described above:

-   -   a) pMet7-HA-Tyk2(C)-LEDGF+pMG2-IN+pXP2d2-rPAPI-luciferase    -   b) pMet7-HA-Tyk2(C)-MDM2+pMG1-p53+pXP2d2-rPAPI-luciferase

Background signal for each Tyk2(C) fusion polypeptide was determined bytransfecting the plasmid that encodes it together with a plasmidencoding an unfused gp130 fragment (pMG1) and the luciferase reporterplasmid (pMet7-HA-Tyk2(C)-LEDGF+pMG1+pXP2d2-rPAPI-luciferase in a);pMet7-HA-Tyk2(C)-MDM2+pMG1+pXP2d2-rPAPI-luciferase in b)). The foldinduction for each tested interaction was calculated as the ratio of theluciferase activity measured relative to the luciferase activitymeasured for the corresponding background signal. The result (FIG. 3)shows that these interactions give rise to strong signals, indicatingthat the method is able to detect interactions in the nucleus.

Example 3 Dose-Dependent Disruption of the Interaction Between Human p53and MDM2 by Nutlin-3

To show that the method can detect modulation of protein-proteininteractions by small molecules, we analyzed the interaction between p53and MDM2, which has been reported to be disrupted by Nutlin-3, a memberof the nutlin family of potential novel anti-cancer compounds (Vassilevet al., 2004). Cells were transfected with the following combinations ofplasmids (100 ng of the Tyk2(C) fusion construct, 1000 ng of the gp130fusion construct and 50 ng of the luciferase reporter construct)according to the methods described above:

-   -   a) pMet7-HA-Tyk2(C)-p53(N)+pMG1+pXP2d2-rPAPI-luciferase    -   b) pMet7-HA-Tyk2(C)-p53(N)+pMG1-EFHA1+pXP2d2-rPAPI-luciferase    -   c) pMet7-HA-Tyk2(C)-p53(N)+pMG2-MDM2+pXP2 d2-rPAPI-luciferase

After transfection, cells were treated with 0-14-41-123-370-1111-3333 nMNutlin-3, final concentration. The results shown in FIG. 4 show adose-dependent decrease of the signal for the interaction between p53(N-terminal region containing the MDM2 binding domain) and MDM2 upontreatment with Nutlin-3, whereas the signal corresponding to theinteraction with EFHA1 (which binds to Tyk2(C) itself) is unaffected bythis treatment. The signal from a control transfection combiningpMet7-HA-Tyk2(C)-p53(N) and the pMG1 plasmid encoding an unfused gp130receptor fragment is low and also unaffected by Nutlin-3 treatment.These data indicate that the method can detect dynamic changes inprotein complexes dependent on addition of exogenous components.

Example 4 Incorporation of Other Kinase Domains in the Method

To support the fact that the method is not limited to the use of thekinase domain derived from Tyk2, we tested fusion proteins comprisingthe kinase domains of Jak2 or c-Src. Similarly to Tyk2, Jak2 belongs tothe Jak family of receptor-associated tyrosine kinases. c-Src belongs tothe Src non-receptor tyrosine kinase family. Cells were transfected withthe following combinations of plasmids (250 ng of the kinase fusionconstruct, 1000 ng of the gp130 fusion construct and 50 ng of theluciferase reporter construct) according to the methods described above:

-   -   a) pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase    -   b) pMet7-HA-Jak2(C)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase    -   c) pMet7-HA-c-Src(K)-RTp66+pMG2-RTp66+pXP2d2-rPAPI-luciferase

Luciferase activity is shown as fold induction relative to theluciferase activity measured in cells transfected with the sameRTp66-kinase domain fusion, an unfused gp130 fragment and the luciferasereporter plasmid (pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase ina); pMet7-HA-Jak2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in b);pMet7-HA-c-Src(K)-RTp66+pMG1+pXP2d2-rPAPI-luciferase in c)). The foldinduction for each tested interaction was calculated as the ratio of theluciferase activity measured relative to the luciferase activitymeasured for the corresponding background signal. The result (FIG. 5)indicates that the interaction between RTp66 subunits can also bedetected using fusion proteins with kinase domains of other kinases thanthat of Tyk2.

Example 5 Inducible Version of the Method

An inducible version of the method was devised to add an additionallevel of control and to allow temporal separation of the proteininteraction and signal generation events. The latter might be importantin cases where modification of the interaction polypeptides by thekinase activity prohibits interaction. A mutant Src tyrosine kinase hasbeen described, the activity of which is made inducible by the smallmolecule imidazole by an arginine to alanine mutation in the catalyticcenter of the enzyme (Qiao et al., 2006). The corresponding pointmutation of this conserved catalytic arginine (R1027) was generated inTyk2, fused to RTp66 and tested in an imidazole-inducible version of theassay. Cells were transfected with the following combinations ofplasmids (250 ng of the kinase fusion construct, 250 ng of the gp130fusion construct and 50 ng of the luciferase reporter construct)according to the methods described above:

-   -   a) pMet7-HA-Tyk2(C)-RTp66+pMG1+pXP2d2-rPAPI-luciferase    -   b) pMet7-HA-Tyk2(C)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase    -   c) pMet7-HA-Tyk2(R1027A)-RTp66+pMG1+pXP2d2-rPAPI-luciferase    -   d)        pMet7-HA-Tyk2(R1027A)-RTp66+pMG2-RTp51+pXP2d2-rPAPI-luciferase

After transfection, cells were treated with 0.5-50 mM imidazole, finalconcentration. The results are represented in FIG. 5, showing animidazole-inducible signal specifically in cells expressing a fusioncontaining the Tyk2 arginine to alanine mutation. Maximalimidazole-dependent induction of the signal, indicative of theinteraction between HIV RTp66 and RTp51, was compared 36-fold tountreated cells. Cells transfected with the Tyk2(C) fusion describedearlier did not exhibit any imidazole regulation. Background signalobserved in cells transfected with combinations of the Tyk2 fusionconstruct and a plasmid encoding an unfused gp130 fragment (pMG1)similarly was unaffected by imidazole treatment. These results indicatethat it is possible to control the interaction assay by applying achemically inducible kinase.

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1. A cytoplasmic protein complex comprising: a first recombinant fusionprotein comprising a kinase fused to a first interaction polypeptide;and a second recombinant fusion protein comprising a domain comprising areporter phosphorylation site, wherein the domain is fused to a secondinteraction polypeptide.
 2. The cytoplasmic protein complex according toclaim 1, wherein the reporter phosphorylation site is phosphorylated. 3.A cytoplasmic protein complex comprising: a first recombinant fusionprotein comprising a mutant kinase fused to a first interactionpolypeptide; and a second recombinant fusion protein comprising a domaincomprising a reporter phosphorylation site, wherein the domain is fusedto a second interaction polypeptide.
 4. The cytoplasmic protein complexaccording to claim 3, wherein the mutant kinase is a mutant constitutivetyrosine kinase.
 5. The cytoplasmic protein complex according to claim3, wherein the mutant kinase is an inactive tyrosine kinase that isactivated by addition of an exogenous small compound.
 6. The cytoplasmicprotein complex according to claim 4, wherein the tyrosine kinase isselected from the group consisting of a Tyk2 mutant, a JAK mutant, and aSrc mutant.
 7. The cytoplasmic protein complex according to claim 1,wherein the first recombinant fusion protein comprises SEQ ID NO:1 orSEQ ID NO:2.
 8. The cytoplasmic protein complex according to claim 1,wherein the reporter phosphorylation domain is comprised in SEQ ID NO:3or SEQ ID NO:4.
 9. A method for detecting a compound-compoundinteraction, the method comprising: fusing a kinase to a firstinteraction polypeptide, resulting in a first recombinant fusion proteinwithout reporter phosphorylation site; fusing a domain comprising areporter phosphorylation site to a second interaction polypeptide,resulting in a second recombinant fusion protein; expressing both thefirst recombinant and second recombinant fusion proteins in a cell andidentifying and/or selecting those cells in which the reporterphosphorylation site is phosphorylated.
 10. The method of claim 9,wherein the cell is a eukaryotic cell.
 11. The method of claim 10,wherein the eukaryotic cell is a mammalian cell.
 12. The methodaccording to claim 9, wherein the kinase is a mutant kinase.
 13. Amethod of detecting a compound-compound interaction, the methodcomprising: fusing a kinase to a first interaction polypeptide, whereinthe mutant kinase is a mutant constitutive tyrosine kinase, resulting ina first recombinant fusion protein without reporter phosphorylationsite; fusing a domain comprising a reporter phosphorylation site to asecond interaction polypeptide, resulting in a second recombinant fusionprotein; expressing both the first recombinant and second recombinantfusion proteins in a cell; and identifying and/or selecting cells inwhich the reporter phosphorylation site is phosphorylated.
 14. Themethod according to claim 12, wherein the mutant kinase is an inactivetyrosine kinase that is activated by addition of an exogenous smallcompound.
 15. The method of claim 13, wherein the constitutive tyrosinekinase is selected from the group consisting of a Tyk2 mutant, a JAKmutant or a Src mutant.
 16. The method according to claim 9, wherein thekinase comprises SEQ ID NO:1 or SEQ ID NO:2.
 17. The method according toclaim 9, wherein the reporter phosphorylation site is comprised in SEQID NO:3 or SEQ ID NO:4.
 18. A cell comprising the cytoplasmic proteincomplex of claim
 1. 19. The cell according to claim 18, wherein the cellis a eukaryotic cell.
 20. The cell of claim 19, wherein the eukaryoticcell is a mammalian cell.
 21. A method for detecting a compound thatdisrupts a polypeptide-polypeptide interaction, the method comprising:growing the cell of claim 18, wherein the cytoplasmic protein complexthereof comprises the polypeptide-polypeptide interaction to bedisrupted, in the absence of and in the presence of at least onecompound; comparing the phosphorylation of the reporter phosphorylationsite in the second recombinant fusion protein of the cytoplasmic proteincomplex of cells, grown in the presence of and in the absence of the atleast one compound; and identifying and/or selecting cells wherein thereporter phosphorylation site is not phosphorylated.